The invention described below relates to an assay procedure for screening potential inhibitors of plant enzymes. In particular, the invention relates to an assay procedure which can be used for the high-throughput screening of potential inhibitors of enzymes of the C4 acid cycle in plants.
The majority of plants can be divided into C3 and C4 plants, depending on the mechanism the plant uses to incorporate CO2 into organic compounds. In C3 plants, CO2 is initially added onto a five-carbon compound forming an unstable six carbon compound which dissociates into two stable three carbon compounds, hence the C3 name. On the other hand, the first stable compound formed in C4 plants is a four carbon compound. An extra biochemical pathway exists in the leaves of C4 plants which allows them to fix CO2 more efficiently and, under certain environmental conditions, to grow more rapidly than their C3 counterparts. In addition, C4 plants use water and nitrogen more efficiently than C3 plants. Together, these properties enable C4 plants to compete favourably with many of the world""s crops, most of which are C3 plants for example, wheat, rice, barley and oats. It follows then that many of the weeds which have an adverse effect on agricultural production throughout the world are C4 plants. Typical examples are nutgrass (Cyperus rotundus), couch grass (Cynodon dactylon), barnyard grass (Echinochloa spp.), Johnson grass (Sorghum halopense), and goose grass (Eleusine indicia). Nutgrass is a particularly serious global problem being present in more than 100 countries and affecting more than 50 crop species. For efficient agricultural production there is an obvious and pressing need for control of C4 weed species. To date, however, no herbicide specific for C4 weeds has been provided.
Crucial to the C4 acid cycle this being the cycle that fixes CO2 in C4 plants are the following enzymes: pyruvate orthophosphate dikinase (pyruvate,Pi dikinase); phosphoenolpyruvate carboxylase; and, NADP-malate dehydrogenase. The pathway involving these enzymes includes the step which incorporates atmospheric CO2 and creates the products which feed into the sugar-producing Calvin cycle. Interruption of this biochemical pathway should, therefore, adversely affect photosynthesis in C4 plants.
Attempts to develop a C4-specific herbicide have involved designing structural analogues of substrates of the C4 acid cycle enzymes. Those exhibiting inhibitory effects have been further modified to maximise their effect. The only report of a compound which specifically inhibited a C4 enzyme related to 3,3-dichloro-2(dihydroxy-phosphinoylmethyl)propenoate which acts on phosphoenolpyruvate carboxylase (see C. L. D. Jenkins et al., Biochem. Int. 14, 219-226 [1987]; H. G. McFadden et al., Aust. J. Chem. 40, 1619-1629 [1987]; C. L. D. Jenkins, Plant Physiol. 89, 1231-1237 [1989]; and, H. G. McFadden et al., Aust. J. Chem. 4, 301-314 [1989]). However, the compound was found to have no effect on the growth of C4 plants. At present, none of the compounds known to inhibit enzymes of the C4 acid cycle has herbicidal activity.
Nevertheless, inhibiting the C4 acid cycle to kill C4 plants remains a promising herbicidal strategy. It has been shown that C4 plants transformed by molecular means (antisense technology) to decrease the level of pyruvate, Pi dikinase or phosphoenolpyruvate carboxylase, enzymes specific to the C4 acid cycle, are incapable of surviving unless grown under high CO2 conditions (see J. P. Maroco et al., Plant Physiol. 116, 823-832 [1998]). Therefore, it follows that a compound that inhibits either pyruvate, Pi dikinase or phosphoenolpyruvate carboxylase might be an efficient and selective herbicide, thus preventing the deleterious effect of C4 weeds on C3 crops.
Marine organisms are an abundant source of compounds of benefit to humans. Many pharmaceuticals are isolated from plants or are derivatives of compounds first identified in marine organisms. Compounds of marine origin are also known which are enzyme inhibitors. Thus, it is reasonable to assume that because of the diversity of the compounds produced by marine organisms there are likely to be some that are inhibitors of C4 acid cycle enzymes.
In the absence of an indication that an organism produces a compound having desired properties, identification of useful compounds in marine organisms usually entails the screening of extracts from thousands of organisms. However, the known assays for the above three C4 enzymes, which are spectrophotometric assays, are large-volume assays and are not suitable for the screening of large numbers of samples. There is thus a need for an assay which can be used to screen large numbers of samples for potential inhibitors of the C4 acid cycle enzymes.
The object of the invention is to provide a high-throughput assay which can be used to screen potential inhibitors of enzymes of the C4 acid cycle in plants.
According to a first embodiment of the invention, there is provided an assay for inhibitors of C4 acid cycle enzymes of plants, the assay comprising:
a) testing for inhibition of at least one of pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including a sample containing the potential inhibitor in a test mixture comprising pyruvate orthophosphate dikinase and substrates thereof, phosphoenolpyruvate carboxylase and the substrate bicarbonate, and malate dehydrogenase and the substrate NADH;
ii) incubating said test mixture under conditions appropriate for the conversion of pyruvate to malate with oxidation of NADH; and
iii) detecting inhibition of at least one of said pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in (a)(ii);
b) testing for inhibition of phosphoenol pyruvate carboxylase or malate dehydrogenase with any sample which contains an inhibitor of at least one of said pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including said sample in a test mixture comprising phosphoenolpyruvate carboxylase and substrates thereof, and malate dehydrogenase and the substrate NADH;
ii) incubating said test mixture under conditions appropriate for the conversion of phosphoenolpyruvate to malate with oxidation of NADH; and
iii) detecting inhibition of said phosphoenolpyruvate carboxylase or malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in (b)(ii);
c) testing for inhibition of malate dehydrogenase with any sample which contains an inhibitor of said phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including said sample in a test mixture comprising malate dehydrogenase, oxaloacetate and NADH or including oxaloacetate in said test mixture from a(ii) or b(ii);
ii) incubating said test mixture under conditions appropriate for the conversion of oxaloacetate to malate with oxidation of NADH; and
iii) detecting inhibition of said malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in (c)(ii).
According to a second embodiment of the invention, there is provided an assay for inhibitors of C4 acid cycle enzymes of plants, the assay comprising:
a) testing for inhibition of at least one of pyruvate orthophosphate dikinase, phospho-enolpyruvate carboxylase or malate dehydrogenase by:
i) including a sample containing the potential inhibitor in a test mixture comprising pyruvate orthophosphate dikinase and substrates thereof, phosphoenolpyruvate carboxylase and the substrate bicarbonate, and malate dehydrogenase and the substrate NADH; and
ii) incubating said test mixture under conditions appropriate for the conversion of pyruvate to malate with oxidation of NADH;
iii) detecting inhibition of at least one of said pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in (a)(ii);
b) testing for inhibition of phosphoenolpyruvate carboxylase or malate dehydrogenase with any sample which contains an inhibitor of at least one of said pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including phosphoenolpyruvate in said test mixture from (a)(ii);
ii) incubating said test mixture under said conditions used in step (a)(ii); and
iii) detecting inhibition of said phosphoenolpyruvate carboxylase or malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in (a)(ii);
c) testing for inhibition of malate dehydrogenase with any sample which contains an inhibitor of phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including oxaloacetate in said test mixture from (a)(ii) or b(ii) or including said sample in a test mixture comprising malate dehydrogenase, oxaloacetate and NADH;
ii) incubating said test mixture under said conditions used in step (a)(ii); and
iii) detecting inhibition of said malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in (a)(ii).
Other aspects of the invention and the best mode of carrying out the invention will become apparent from a reading of the following detailed description.
The following abbreviations are used hereafter:
ATP adenosine triphosphate
AMP adenosine monophosphate
Pi inorganic phosphate
PPi inorganic pyrophosphate
NADH nicotinamide-adenine dinucleotide (reduced form)
NAD+ nicotinamide-adenine dinucleotide (oxidised form)
PPDK pyruvate orthophosphate dikinase (EC 2.7.9.1)
PEP C phosphoenolpyruvate carboxylase (EC 4.1.1.31)
MDH NAD-malate dehydrogenase (EC 1.1.1.37)
HEPES N-[2-hydroxyethyl]piperazine-Nxe2x80x2-[2-ethanesulfonic acid]
OAA Oxaloacetate
The term xe2x80x9ccomprisingxe2x80x9d as used in the above definition of the embodiments and hereafter denotes a mixture or composition which includes at least the specified components but should not be interpreted as meaning that the mixture or composition consists of only those components. Mixtures and compositions can include other ingredients known to those of skill in the art as normal components of mixtures for the assay of enzymes. Examples of these other ingredients include buffers with a suitable pKa to allow control of the assay mixture within the desired pH range such as HEPES, 2-(N-Morpholino)ethanesulphonic acid (MES), 3-N-Morpholino)propanesulphonic acid (MOPS) or Tris(hydroxymethyl)aminomethane (Tris) but excluding phosphate buffers which will interfere with the reaction; reducing agents such as dithiothreitol or xcex2-mercaptoethanol and salts such as the salts of mono and divalent options. For example PPDK is a divalent cation dependent enzyme and requires the presence of a divalent cation, such as Mg2+, Ca2+ and Mn2+. PEP C is also divalent cation dependent, being dependent on the presence of a divalent metal cation. Other salts, such as ammonium sulphate may be added to improve the performance of the assay.
The present inventors have found that the activities of the C4 plant enzymes PPDK and PEP C can conveniently be assayed in a microtitre plate format using the procedures summarised above thus allowing for the screening of a large number of potential inhibitors of these enzymes or of samples suspected of containing inhibitors. The reactions involved in the assay are as follows: 
in which the abbreviations set out above have been used. The MDH used for the final reaction in the above sequence is preferably not the C4 plant NADP-dependent enzyme, but is instead the ubiquitous NAD-dependent enzyme. Use of the ubiquitous NAD-dependent enzyme in assays allows elimination of inhibitors that might have a general effect on plants in not being specific for C4 plant enzymes.
As NADH is a substrate for the enzyme catalysing the final reaction in the sequence, the progress of the reaction can be measured via the oxidation of NADH which has a maximal absorbance at 340 nm (range, 300-400 nm). Equally, the reaction can be monitored for the production of NAD+ by the maximal absorbance of NAD+ at approximately 260 nm (range, 230-290 nm). If the latter approach is taken, microtitre plates which have little or no UV absorbing properties are required.
With reference to the assay according to the first embodiment of the invention, step (a) of this assay constitutes a primary screening procedure for compounds active against C4 plant enzymes. Samples identified as containing an active constituent that is, a constituent that reduces the amount of NAD+ formed and thus inhibits at least one of PPDK, PEP C or MDH are then subjected to a secondary screening to determine the enzyme specificity of the inhibitor. The secondary screening is done by assaying individual enzymes in accordance with steps (b) and (c) of the first embodiment and/or by carrying out a physiological assay in accordance with the second embodiment of the invention.
As indicated above, assays can be performed in 96-well microtitre plates. Plates can have wells with round or flat bottoms and can be made of any material that does not have an appreciable absorbance at the wavelength to be measured (260 or 340 nm). The usual volume of a reaction mixture is about 100 xcexcl but this can be adjusted to suit the capacity of the microtitre plate. All that is required is that the absorbance value initially attained is significantly different to the background absorbance of the microtitre plate plus the absorbance of the assay mixture minus NADH or NAD+.
A general description of the method of carrying out a primary screening that is, a screening using the assay according to step (a) of the first embodiment of the invention follows. Typical components of mixtures for this assay and the concentrations of components are set out in Table I
With regard to the enzymes, assay mixtures typically comprise the following: PPDK, 0.005 units; PEC C; 1 unit; and, MDH, 2 units.
Although a pH of 8.0 is given in Table I above, assays can be performed within a pH range of 6.0 to 9.5.
All mixture components save for at least one of the PPDK substrates are added to wells of a microtitre plate. In addition to test wells to which the test sample is added, positive and negative control wells are also prepared. Positive controls include all assay components save for the test sample. However, an equal volume of test sample solvent (for example, water, methanol, ethanol, DMSO, or mixtures thereof) is added to positive controls. Both test sample and substrate used to initiate the reaction are omitted from the negative control but suitable volumes of the appropriate solvent are added in place of these components. Test wells are typically prepared in duplicate but samples can be tested singly or in greater than two replicates.
After all components have been added to the test and control wells, the plate is usually allowed to equilibrate at room temperature (20-30xc2x0 C.) for 5 minutes to allow dispersion of test samples and control solvent through the reaction mixtures to give a consistent absorbance reading. This equilibration can be for as little as 1 minute but can also be for up to several hours as the enzymes and other assay components are stable. After equilibration, the absorbance at 340 nm (or 260 nm) is measured using a spectrophotometric plate reader. This measurement is defined as the initial absorbance, A0. The reaction is started by adding, to all but the negative control wells, the substrate pyruvate to the desired final concentration. Alternatively, the test and positive control assay mixtures can be made with pyruvate and without ATP. The reaction is then initiated with ATP.
Plates are incubated at 20 to 30xc2x0 C. until the reaction is complete as evidenced by a constant reading for the positive controls. This is usually reached after 10 to 60 minutes depending on the amount and activity of the enzymes. The absorbance is then again measured at 340 nm (or 260 nm) to determine the final absorbance, Aend. To obtain a percent inhibition for an extract being tested, the difference between A0 and Aend for the test sample is standardised against the difference between A0 and Aend for the positive control.
In the individual enzyme approach to secondary screening of sample extracts having inhibitory activity, the first step is to test active samples against PEP C and MDH. This is step (b) of the first embodiment defined above. Typical components of a PEP C/MDH assay mixture are set out in the following table.
The PEP C/MDH assay is carried out in essentially the same manner as the primary screening assay. If the reaction is not inhibited, specific inhibition of PPDK by active extracts from the primary screen is indicated.
If inhibition is observed in the PEP C and MDH assay, a further assay is conducted in which active extracts are tested against MDH alone. This second step of the secondary screening corresponds to step (c) of the first embodiment. Typical components of an MDH assay mixture are set out in the following table.
If the MDH reaction is not inhibited, the step 1 result can be interpreted as indicating that the inhibitor acts on either PEP C only, or PEP C and PPDK, but not on MDH.
Active extracts identified in the primary screen can also be tested using the physiological assay according to the second embodiment. Inhibition of PPDK is first assessed with the physiological assay using the components listed above in Table I and using essentially the same procedure as for the primary screen. Inhibition of PEP C is then tested by adding the substrate, phosphoenol pyruvate, to the reaction mixture. If an inhibitor is specific for PPDK, no inhibition of PEP C is seen at this stage for the extract containing such an inhibitor. If, however, inhibition is seen, the MDH substrate oxaloacetate is added and the effect of the inhibitor on this enzyme assessed. As with the isolated enzyme approach, normal MDH activity indicates that the inhibitor acts on either PEP C only, or PEP C and PPDK, but not on MDH.
With the assays described above, compounds, or at least extracts containing such compounds, can be identified that are specific inhibitors of the C4 plant enzymes, PPDK and PEP C. The ability to differentiate between inhibitors of these enzymes and inhibitors of MDH allows elimination of those compounds that also act on the more catholic enzyme MDH and thus would not be useful as specific C4 plant herbicides.
It should be noted that while it is not necessary to subject samples which show no inhibition in step a) to further steps b) and c), or to subject samples inactive in step b) to step c), it may be convenient, especially when performing a high throughput assay, to subject all samples to steps a), b) and c) regardless of whether inhibition is seen in steps a) and b). Subjecting all samples to all steps may serve as a useful cross check.
The assays according to the invention can be used to screen for potential inhibitors derived from any source including plants, animals, bacteria, fungi and protozoans. Crude aqueous or organic extracts can be tested, typical organic solvents being methanol, ethanol, dimethylsulfoxide (DMSO) or any combination thereof. Extracts can be acidic or basic provided that any alteration of pH of reaction mixtures on addition of extract does not interfere with the progress of reactions. Compounds able to be tested in the assays include any isolated natural or synthetic product, or any combination of compounds whether produced by combinatorial, purification, or synthetic processes.
The C4 plant enzymes PPDK and PEP C can be prepared by any of the methods known to those of skill in the art. For example, Chapter 3 of Methods in Plant Biochemistry, Vol. 3 (Academic Press Limited, London, England, 1990, pp. 39-72), which is by Anthony R. Ashton et al. and is entitled xe2x80x9cEnzymes of C4 Photosynthesisxe2x80x9d, includes summaries of methods for the purification of PPDK and PEP C. The entire content of Chapter 3 of this volume is incorporated herein by cross-reference. MDH is widely available from commercial sources.
Having broadly described the assays according to the invention, the results of application of the assays to the screening of extracts of marine organisms for inhibitors of PPDK and PEP C will now be provided as a non-limiting example of the invention.